The mechanism of replication initiation of the mitochondrial DNA (mtDNA) in the yeast Saccharomyces cerevisiae is poorly understood, in part because a high rate of recombination has made it difficult to use common physical approaches. However, the accumulated evidence suggests that two mechanisms of replication initiation may exist. One, analogous to initiation in mammalian mtDNA, may employ a promoter and transcript cleavage to generate a primer. The other mechanism that operates in the absence of transcription, may initiate replication forks through end/internal recombination events. We will examine these mechanisms using a combination of structural and genetic approaches. A new 2-D gel electrophoresis technique will be used to systematically search the mitochondrial genome for various possible initiation structures. Mutants with a temperature sensitive defect in the initiation of mtDNA replication will be isolated and characterized. Double mutants defective in transcription and in possible recombination pathways will be examined for failure to maintain mtDNA. A mutant that abolishes the biased inheritance of a class of mitochondrial deletion genomes, thought to have a replication advantage because of a high concentration of replication origins, will be studied.